Obesity affects a majority of American adults, with diverse and significant detrimental effects on human health. Despite a major role for genetic background in obesity, only a small number of the human genes that predispose individuals to obesity have been identified. Understanding the pathways that control storage of body fat will be crucial for pinpointing genes likely to cause susceptibility to this disease. The long-term goal is to identify genes whose activities can be modified to prevent or treat human obesity. The goals of this application are to elucidate the mechanism by which the related RNA-binding proteins Spen and Nito regulate adiposity, and to identify other candidates for cell-autonomous regulation of adiposity. A fruit fly model has been developed to dissect the tissue specificity of gene function in the regulation of body fat levels, as well as new tools to parse out the contributions of behavioral alterations and to directly measure rates of fat incorporation into stores. A complementary approach using cultured cells will directly translate findings in the fly model to functions in mammalian fat storage. The central hypothesis is that genes acting autonomously in the fruit fly fat-storage tissue (the fat body, FB) to control levels of body fat will play conserved roles in mammalian fat storage. This idea is supported by the applicant's previous success in identifying such genes, and by preliminary data analyzing specific candidate genes, like Spen. The rationale for this project is that regulatory pathways in fat storage tissues must respond to organismal cues to control levels of stored fat, and that identifying key genes acting in these pathways may translate directly to insights into genetic predispositions to human obesity. This model will be tested by pursuing three specific aims: (1) Test the hypothesis that Spen and Nito function in an opposing manner to regulate body fat. (2) Test the hypothesis that Spen/Nito regulate energy balance by binding specific RNAs to alter gene expression; and (3) Identify candidate genes for novel, conserved autonomous regulators of fat storage. In Aim 1, we will determine the mechanistic basis of defects leading to altered fat in fly larvae lacking Spen and/or Nito, two RNA-binding proteins in the same family known to modulate transcriptional output of other pathways but never before implicated in the control of adiposity. In Aim 2, we test a model that Spen and Nito bind specific RNAs to target specific metabolic target genes for transcriptional control. In the Aim 3, the mouse orthologs of fly genes that directly regulate fat storage in the FB (including Shep, Rala and NFAT) will be analyzed functionally in cultured mouse adipocytes to identify those that also control mammalian fat storage in an autonomous manner. This innovative combination of approaches will uncover new roles for genes whose functions in fat regulation are currently unknown. The significance of this proposal lies in its potential to elucidate a new pathway controlling fat storage via RNA-binding proteins, and to characterize other candidate obesity genes, providing significant insights into the multigenic nature of this disease, and identifying new targets for future treatments.